microorganisms Review Deciphering Streptococcal Biofilms 1, 1, 2, 1 3 Puja Yadav * , Shalini Verma y, Richard Bauer y, Monika Kumari , Meenakshi Dua , Atul Kumar Johri 4, Vikas Yadav 4 and Barbara Spellerberg 2,* 1 Department of Microbiology, Central University of Haryana, Mahendergarh, Haryana 123031, India; [email protected] (S.V.); [email protected] (M.K.) 2 Institute of Medical Microbiology and Hygiene, University Hospital Ulm, 89073 Ulm, Germany; [email protected] 3 School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110067, India; [email protected] 4 School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India; [email protected] (A.K.J.); [email protected] (V.Y.) * Correspondence: [email protected] (P.Y.); [email protected] (B.S.) These authors contributed equally to this work. y Received: 19 October 2020; Accepted: 17 November 2020; Published: 21 November 2020 Abstract: Streptococci are a diverse group of bacteria, which are mostly commensals but also cause a considerable proportion of life-threatening infections. They colonize many different host niches such as the oral cavity, the respiratory, gastrointestinal, and urogenital tract. While these host compartments impose different environmental conditions, many streptococci form biofilms on mucosal membranes facilitating their prolonged survival. In response to environmental conditions or stimuli, bacteria experience profound physiologic and metabolic changes during biofilm formation. While investigating bacterial cells under planktonic and biofilm conditions, various genes have been identified that are important for the initial step of biofilm formation. Expression patterns of these genes during the transition from planktonic to biofilm growth suggest a highly regulated and complex process. Biofilms as a bacterial survival strategy allow evasion of host immunity and protection against antibiotic therapy. However, the exact mechanisms by which biofilm-associated bacteria cause disease are poorly understood. Therefore, advanced molecular techniques are employed to identify gene(s) or protein(s) as targets for the development of antibiofilm therapeutic approaches. We review our current understanding of biofilm formation in different streptococci and how biofilm production may alter virulence-associated characteristics of these species. In addition, we have summarized the role of surface proteins especially pili proteins in biofilm formation. This review will provide an overview of strategies which may be exploited for developing novel approaches against biofilm-related streptococcal infections. Keywords: streptococci; opportunistic pathogen; planktonic; biofilm; antibiotic therapy; quorum sensing (QS) 1. Introduction Biofilms are surface-associated microbial communities enclosed within a self-produced matrix consisting of a single or multiple bacterial species [1]. Following the evolution of prokaryotes several billion years ago, the evolution of biofilms is considered a defense mechanism against harsh environmental conditions by providing homeostasis and protection to the involved bacterial cells [2]. Biofilms were first observed on tooth surfaces by Antoni van Leeuwenhoek in the 17th century, while the term “biofilm” was introduced into medical microbiology by Costerton in 1982 [3]. He reported biofilm formation of S. aureus on an infected endocardial pacemaker. Microorganisms 2020, 8, 1835; doi:10.3390/microorganisms8111835 www.mdpi.com/journal/microorganisms Microorganisms 2020, 8, 1835 2 of 31 Biofilms play a prominent role in human infections. More than 80% of microbial diseases have been linked to biofilm formation. Bacterial biofilms are involved in infections of the urinary tract, the female genital tract, the bloodstream, and the upper respiratory tract [4–6]. Dental plaque, which contains streptococci and may eventually result in caries, is a prime example of a natural biofilm composed of a multispecies bacterial community. In biofilms, increased resistance to or tolerance of antibiotics is a common problem. It may be intrinsicMicroorganisms due to the 2020 microbial, 8, x FOR PEER growth REVIEW conditions or it may be caused by mutations or the exchange3 of 34 of antibiotic resistance genes [7,8]. As biofilms adapt to survive antibiotic treatment, infections are hard to eradicate2.2. Nucleic despite Acids proper antibiotic therapy [6,9,10]. Most streptococcalExtracellular DNA species (eDNA) reside is a asnother commensals major component on mucous of the membranes, biofilm matrix while. It is highly several similar pathogenic streptococcito the genomic are responsible DNA of the for bacterial life-threatening species present human within infections. the biofilm Theand is production released through of biofilms is a commonbacterial phenotypecell lysis [26] of. It commensal plays a role asin welladhesion as pathogenicand is essential species. in biofilm Commensal stabilization streptococci and biofilmsmaintenance. represent theirFurthermore, natural eDNA lifeform, provides and inprotection pathogenic against streptococcal antimicrobial species, peptides biofilms and divalent have been cations through chelation of these substances [26]. Upon the addition of nucleases to streptococcal identified as important determinants of infectious diseases. This review will focus on specifics of biofilms, significant inhibitory and disintegrating effects on biofilm formation have been observed streptococcalfor S. pneumonia, biofilms, S. theirpyogene regulation,s, as well as for and viridans the potential streptococci to interfere[27–29]. with biofilm production as a therapeutic approach. 2.3. Extracellular Proteins 2. Biofilm Composition The third major component in biofilm matrix, are extracellular proteins. Extracellular proteins Thefacilitate development reorganization, of these degradation, highly ordered andmulticellular dispersal of the communities biofilm matrix is a and complex play a multistep structural process role [11] (Figurein1 ).biofilm Durings [30] biofilm. A common formation, theme thefor several bacterial biofilm cells associated transform proteins from planktonic of Gram-positive life to bacteria an immotile life formis their [1,12 ability], resulting to form in amyloids. a complex These community include BAP consisting of S. aureus of, diEPSfferent of Enterococci, microbial and subpopulations. P1 of S. mutans [31–33]. Amyloid proteins, which assemble into insoluble fibrils, participate supportively in Biofilm formation is initiated with the adhesion of planktonic bacteria to biotic or abiotic surfaces, the cell aggregation and in biofilm formation [34]. Another form of fibrillar proteins are streptococcal developmentpili, which of microcolonies,are highly structured and cell a successive surface appendages production consisting of an extracellularof several differe matrixnt structural composed of polymericproteins. substances Their special such asrole proteins, in the formation polysaccharides, of biofilms and will extracellular be discussed DNA in a later [13]. section Three-dimensional of this structuresreview. develop The biofilm through matrix maturation however, andalso contains finally result nonfibrillar in the proteins detachment like, e.g., of singlethe Glucan bacterial-binding cells [14]. Microbialproteins cells (Gbps) in biofilm in S. mutans experience, which impaired play a signifi diffcantusion role of for nutrients biofilm formation and waste as productsthey promote with less nutrientaggregation availability and in plaque the core cohesion of the [35,36] biofilm. Furthermore [15]. In addition,, some of the due proteins to increased within the endogenous biofilm matrix oxidative stress withinare enzymes biofilms, involved microbial in the degradation cells are subjected of EPS and to spontaneousthe initiation of mutations a new biofilm [16]. lifecycleGenetic. The variations degradation of biopolymers also delivers energy and carbon sources to bacterial biofilm cells, then give rise to microbial subpopulations that are physiologically heterogeneous [17,18]. especially under limited nutrient availability [37]. FigureFigure 1. Schematic 1. Schematic diagram diagram representing representing the lifethe cyclelife cycle of biofilm of biofilm formation formation in Streptococci. in Streptococci. The The diagram showsdia thegram transition shows of the planktonic transition cellsof planktonic to sessile cells cells to by sessile undergoing cells by diundergoingfferent stages different of biofilm stages formation of and repeatingbiofilm formation the cycle and by therepeating conversion the cycle of by sessile the conversion cells to the of planktonicsessile cells stateto the again. planktonic state again. Microorganisms 2020, 8, 1835 3 of 31 The biofilm matrix is essential for protection of bacteria from environmental stresses and consists of extracellular polymeric substances (EPS). It represents an efficient diffusion barrier, interfering with the penetration of harmful substances inside the biofilm [1]. Differential gene expression is responsible for the production of EPS, which provides cohesion of the bacterial cells and determines the structure of the biofilm. Three major components are found in the biofilm matrix in varying amounts: extracellular polysaccharides, extracellular nucleic acids, and proteins can be detected together with a high percentage of water. 2.1. Extracellular Polysaccharide Microscopic evaluation of biofilms shows exopolysaccharides as elongated or branched filaments mediating the adhesion